Barry M. Markaverich

Bioflavonoid interaction with rat uterine type ii binding sites and cell growth inhibition

Competition analysis with a number of known bioflavonoids demonstrated that these compounds (luteolin, quercetin, pelargonin) compete for [3H]estradiol binding to cytosol and nuclear type II sites in rat uterine preparations. The inhibition of [3H]estradiol binding to type II sites was specific and these bioflavonoids did not interact with the rat uterine estrogen receptor. Since estradiol stimulation of nuclear type II sites in the rat uterus is highly correlated with cellular hypertrophy and hyperplasia, we assessed the effects of these compounds on the growth of MCF-7 human breast cancer cells in culture and on estradiol stimulation of uterine growth in the immature rat. The data demonstrated that addition of quercetin (5-10 micrograms/ml) to MCF-7 cell cultures resulted in a dose-dependent inhibition of cell growth (DNA/flask). This effect was reversible by removal of quercetin from the culture medium, or by the addition of 10 nM estradiol-17 beta to these cell cultures containing this bioflavonoid. Since estradiol-17 beta (10 nM) stimulated nuclear type II sites and proliferation of MCF-7 cells, we believe bioflavonoid inhibition of MCF-7 cell growth may be mediated through an interaction with nuclear type II sites. This hypothesis was confirmed by in vivo studies which demonstrated that injection of luteolin or quercetin blocked estradiol stimulation of nuclear type II sites in the immature rat uterus and this correlated with an inhibition of uterine growth (wet and dry weight). These studies suggest bioflavonoids, through an interaction with type II sites, may be involved in cell growth regulation.

Progesterone and dexamethasone antagonism of uterine growth: A role for a second nuclear binding site for estradiol in estrogen action

Abstract Administration of estradiol to mature-ovariectomized rats caused nuclear binding of the estrogen receptor and elevated the number of secondary estradiol binding sites (type II sites) in uterine nuclei. These events are correlated with the stimulation of uterine growth. Administration of dexamethasone or progesterone blocks the stimulation of nuclear type II sites and inhibits uterine growth. The inhibition of growth occurs even though the estrogen receptor undergoes normal nuclear translocation, nuclear retention and cytoplasmic replenishment. It is speculated that the stimulation of nuclear type II sites may be an intermediate step in the mechanism by which estradiol causes uterine growth.

Effects of estradiol-17α on nuclear occupancy of the estrogen receptor, stimulation of nuclear type II sites and uterine growth

Estriol and estradiol-17 alpha (E2-17 alpha) have classically been described as weak or impeded estrogens since they are incapable of stimulating true uterine growth when administered acutely by single injection. We have demonstrated [16] that estriol is capable of stimulating true uterine growth when the hormone is administered by paraffin implant. The possibility that E2-17 alpha is similar to estriol was examined. A single injection of E2-17 alpha causes a rapid accumulation of the estrogen receptor in uterine nuclei and this is correlated with the stimulation of early uterotropic responses. The nuclear receptor content declines rapidly and no stimulation of nuclear type II sites or true uterine growth is observed. E2-17 alpha does however stimulate the replenishment of cytoplasmic estrogen receptor. This receptor-response profile is typical of a short acting estrogen such as estriol. Chronic exposure (96 h) of mature-ovariectomized rats to estradiol-17 alpha (4 mg) by beeswax implant results in continual nuclear occupancy by estrogen receptors, dramatic stimulation of nuclear type II sites and true uterine growth. It is not possible to determine whether the uterotropic stimulation was due to direct effects of E2-17 alpha since this isomer was partially metabolized to E2-17 beta and both isomers were found in uterine nuclei after an implant of E2-17 alpha. We conclude that E2-17 alpha is capable of acting as an estrogen, either by its inherent estrogenicity or by its conversion to E2-17 beta, and that it may be dangerous to consider this steroid to be an ineffective or inadequate estrogen.

Heterogeneity of Estrogen Binding Sites: Relationship to Estrogen Receptors and Estrogen Responses

Publisher Summary This chapter discusses the significance of the various forms of estrogen binding sites. Estrogen receptors are macromolecules that bind estrogen to form receptor hormone complexes. These complexes are generally involved with the stimulation of cellular growth and metabolism. This concept of steroid hormone action is widely known. Saturation analysis of uterine cytosol over a wide range of [ 3 H] estradiol concentrations reveals a curve for specifically bound [ 3 H] estradiol. Two types of macro molecule exist that bind estrogen in a stereospecific fashion: (1) type I, which has the properties of the classical cytosol estrogen receptor, and (2) type II, which has a lower affinity and a higher capacity for estradiol than type I. Type II sites also differ from type I as they do not undergo translocation from the cytoplasm to the nucleus after an estrogen injection. The implications of the existence of type II sites are far-reaching. Their presence interferes with the measurement of type I sites, producing an overestimate of type I and an incorrect identification of type II as type I.

The agonistic and antagonistic actions of estriol

Estriol is a short acting estrogen, and as such, displays both agonistic and antagonistic properties, when it is injected in saline solution. These are predictable attributes of any short acting agonist if one considers the basic pharmacology of receptor ligand binding interactions. That is, short acting hormones or drugs occupy receptor sites for protracted time periods and, although they stimulate responses, these are of short duration. However, when estriol is administered in continuous fashion the estrogen receptor is occupied for long periods of time and full estrogenic responses are observed, and there is no antagonism of estradiol action. The purpose of this paper is to review the work that leads to the above conclusion and to discuss our more recent work on the relationship between estrogen receptor binding and the stimulation of type II estrogen binding sites.

Estrogen binding in the rat uterus: heterogeneity of sites and relation to uterotrophic response.

Abstract Multiple species of estrogen binding sites have been demonstrated in cytosol and nuclear fractions of uterine cells from immature and adult ovariectomized rats. Cytosol . Type I sites show properties identical to the classical cytoplasmic estrogen receptor. Type II binding sites have lower affinity for the ligand, exist in higher concentrations and do not undergo nuclear translocation after estrogen stimulation. These secondary sites may be involved in the retention of estrogens within the uterus, by creating an estrogen rich environment for binding to type I sites which in turn translocate estrogen as a receptor-hormone complex to the nucleus. Since it is not known whether type II sites are intra- or extracellular, they may act in this buffer capacity in either or both cellular compartments. Another possibility is that type II sites are precursors of type I sites. Nucleus . In addition to type I sites, a second site was also observed in the nuclear compartment. This second nuclear binder does not appear to be related to the cytosol type II site; however they have similar dissociation constant and share hormone and tissue specificity. Estradiol administration causes an increase in both specific binding components in uterine nuclei of mature ovariectomized rats. The second component (type II) binds estradiol with a higher capacity than type I sites, and displays a saturation curve which is sigmoidal. The stimulation of type II sites is a specific estrogenic response and is highly correlated with uterine growth. A single injection of estradiol results in long term retention of type I sites (6h), rapid and sustained elevations of type II sites (1–72 h) and true uterine growth. In contrast, estriol injection caused a rapid increase in type I sites which was not accompanied by an increase in type II sites and no true uterine growth occurred. Conversely the administration of estriol by paraffin implant sustained elevated nuclear levels of type I sites, increased nuclear type II sites 2–3 fold above controls and stimulated true uterine growth 48 h following hormone administration. These data suggest that estrogen stimulation of true uterine growth may require long term (6–24 h) nuclear retention of type I sites and sustained elevation in type II sites. These secondary sites do not appear to be translocated to the nucleus but instead may be chromosomal proteins which are present in the nucleus of uterine cells at alt times. We conclude that elevated levels of nuclear type II sites correlate well with the biosynthetic events associated with the long term uterotrophic response to estrogenic hormones.

Leukotoxin Diols from Ground Corncob Bedding Disrupt Estrous Cyclicity in Rats and Stimulate MCF-7 Breast Cancer Cell Proliferation

Previous studies in our laboratory demonstrated that high-performance liquid chromatography (HPLC) analysis of ground corncob bedding extracts characterized two components (peak I and peak II) that disrupted endocrine function in male and female rats and stimulated breast and prostate cancer cell proliferation in vitro and in vivo. The active substances in peak I were identified as an isomeric mixture of 9,12-oxy-10,13-dihydroxyoctadecanoic acid and 10,13-oxy-9,12-dihydroxyoctadecanoic acid, collectively designated tetrahydrofurandiols (THF-diols). Studies presented here describe the purification and identification of the HPLC peak II component as 9,10-dihydroxy-12-octadecenoic acid (leukotoxin diol; LTX-diol), a well-known leukotoxin. A synthetic mixture of LTX-diol and 12,13-dihydroxy-9-octadecenoic acid (isoleukotoxin diol; i-LTX-diol) isomers was separated by HPLC, and each isomer stimulated (p < 0.001) MCF-7 cell proliferation in an equivalent fashion. The LTX-diol isomers failed to compete for [3H]estradiol binding to the estrogen receptor or nuclear type II sites, even though oral administration of very low doses of these compounds (>> 0.8 mg/kg body weight/day) disrupted estrous cyclicity in female rats. The LTX-diols did not disrupt male sexual behavior, suggesting that sex differences exist in response to these endocrine-disruptive agents.

Tetrahydrofurandiols (THF-diols), Leukotoxindiols (LTX-diols), and Endocrine Disruption in Rats

Background Ground corncob animal bedding and corn food products contain substances that disrupt endocrine function in rats. The disruptors were identified as isomeric mixtures of tetrahydrofurandiols (THF-diols; 9,12-oxy-10,13-dihydroxyoctadecanoic acid and 10,13-oxy-9,12-dihydroxyoctadecanoic acid) and leukotoxindiols (LTX-diols; 9,10-dihydroxy-12-octadecenoic acid and 12,13-dihydroxy-9-octadecenoic acid). The authentic compounds blocked sexual behavior in male rats and estrous cyclicity in female rats at oral doses of 2 ppm. Objectives To define the lowest observed adverse effect level (LOAEL) for the THF-diols and LTX-diols in rats, we examined the nature of their interaction (additive or synergistic) and quantified the concentration of THF-diols in rat tissues. Methods Adult male and female rats were provided drinking solutions containing various doses of THF-diols and/or LTX-diols, and we evaluated their effects on male sexual behavior and female estrous cyclicity. Tissues were collected for THF-diol determination by gas chromatography–mass spectrometry. Results The LOAEL for THF-diols and LTX-diols for blocking estrous cyclicity was 0.5–1.0 ppm and 0.2–0.5 ppm, respectively. Higher concentrations (1–2 ppm) of THF-diols were required to block male sexual behavior. Combination studies with subthreshold doses of 0.05 ppm THF-diols plus 0.05 ppm LTX-diols revealed that their effects on estrous cyclicity were not synergistic. We were unable to detect THF-diols in tissues from rats treated with 10 ppm of the compounds, suggesting that metabolism may be involved. Discussion THF-diols, LTX-diols, and/or their metabolites likely act additively to disrupt endocrine function in male and female rats at concentrations (0.5–1 ppm) that are 200-fold lower than those of classical phytoestrogen endocrine disruptors.

Disruption of male sexual behavior in rats by tetrahydrofurandiols (THF-diols)

The mitogenic agent that disrupts male and female sexual behavior has been isolated from corncob bedding. The disrupting activity resides in an isomeric mixture of linoleic acid derivatives with a tetrahydrofuran ring and two hydroxyl groups (THF-diols) that include 9, (12)-oxy-10, 13-dihydroxtstearic acid and 10, (13)-oxy-9, 12-dihydroxystearic acid. We examined the effects of exposure of male rats to THF-diols in drinking water on several parameters of male sexual behavior. THF-diols disrupt sexual behavior in male rats by reducing mounting and intromission frequencies. The mount, intromission and ejaculatory latencies are enhanced while the ejaculatory responses are diminished. These findings suggest that the THF-diols modulate hypothalamo-pituitary axis to regulate steroid hormone-dependent male sexual behavior.

Cytosol type II sites in the rat uterus: Interaction with an endogenous ligand

Previous studies from our laboratory demonstrated that normal, but not malignant tissues, contain a ligand which competes for [3H]estradiol binding to nuclear type II sites in the rat uterus. Since elevated nuclear levels of type II sites are correlated with estrogen stimulation of uterine growth and DNA synthesis, we believe this ligand may regulate cell growth. The present studies show that the ligand for nuclear type II sites also interacts with type II sites in uterine cytosol. This was demonstrated by dilution experiments which show that greater quantities of type II sites are measured in dilute (10 mg/ml) than in concentrated (40 mg/ml) uterine cytosol. Furthermore, stripping of uterine cytosol with 1% dextrancoated charcoal, or pre-binding cytosol type II sites to hydroxylapetite (HAP) prior to binding analysis, removed the ligand from these preparations such that high levels of type II sites were measured. Following charcoal stripping, cytosol type II sites demonstrated good specificity for estrogenic hormones but not progesterone, corticosterone, or the triphenylethylene anti-estrogen, nafoxidine. Since the level of type II sites in the cytosol always preceded and exceeded the level of this site measured in uterine nuclei at all times following estrogen treatment (0-96 h), we believe cytosol type II sites may function as an type II-ligand binding protein (LBP) which regulates the availability of the ligand for interaction with nuclear type II sites. This is consistent with our observation that type II sites are not depleted from uterine cytosol by estrogen treatment and nuclear type II sites are very tightly associated with the nuclear matrix.